Life Sciences 84 (2009) 713–718
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Enhanced vascular contractility in alpha1-adrenergic receptor-deficient mice Atsushi Sanbe a,⁎,1, Yoshio Tanaka b,1, Yoko Fujiwara a,1, Noriko Miyauchi a, Reiko Mizutani a, Junji Yamauchi a, Susanna Cotecchia c, Katsuo Koike b, Gozoh Tsujimoto d, Akito Tanoue a a
Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo, Japan Department of Chemical Pharmacology, Toho University School of Pharmaceutical Science, Chiba, Japan Institut de Pharmacologie et de Toxicologie, Faculte de Medecine, Lausanne, Switzerland d Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan b c
a r t i c l e
i n f o
Article history: Received 1 September 2008 Accepted 12 February 2009 Keywords: α1-Adrenergic receptor Serotonin Prostaglandin F2α Pressor response
a b s t r a c t Aim: Alpha1-adrenergic receptors (α1-ARs) are classified into three subtypes: α1A-AR, α1B-AR, and α1D-AR. Triple disruption of α1A-AR, α1B-AR, and α1D-AR genes results in hypotension and produces no contractile response of the thoracic aorta to noradrenalin. Presently, we characterized vascular contractility against other vasoconstrictors, such as potassium, prostaglandin F2alpha (PGF2α) and 5-hydroxytryptamine (5-HT), in α1A-AR, α1B-AR, and α1D-AR triple knockout (α1-AR triple KO) mice. Main methods: The contractile responses to the stimulation with vasoconstrictors were studied using isolated thoracic aorta. Key findings: As a result, the phasic and tonic contraction induced by a high concentration of potassium (20 mM) was enhanced in the isolated thoracic aorta of α1-AR triple KO mice compared with that of wild-type (WT) mice. In addition, vascular responses to PGF2α and 5-HT were also enhanced in the isolated thoracic aorta of α1-AR triple KO mice compared with WT mice. Similar to in vitro findings with isolated thoracic aorta, in vivo pressor responses to PGF2α were enhanced in α1-AR triple KO mice. Real-time reverse transcription-polymerase chain reaction analysis and western blot analysis indicate that gene expression of the 5-hydroxytryptamine 2A (5-HT2A) receptor was up-regulated in the thoracic aorta of α1-AR triple KO mice while the prostaglandin F2α receptor (FP) was unchanged. Significance: These results suggest that loss of α1-ARs can lead to enhancement of vascular responsiveness to the vasoconstrictors and may imply that α1-ARs and the subsequent signaling regulate the vascular responsiveness to other stimulations such as depolarization, 5-HT and PGF2α. © 2009 Elsevier Inc. All rights reserved.
Introduction Alpha1-adrenergic receptors (α1-ARs) are mediators of catecholamines such as noradrenaline (NA) and adrenaline, and these receptors function as regulators of various physiological functions such as smooth muscle contraction, blood pressure (Sanbe et al. 2007; Tanoue et al. 2002), cardiac growth (O'Connell et al. 2003), and glucose homeostasis (Burcelin et al. 2004). The α1-ARs have been classified into three receptor subtypes, α1A-AR, α1B-AR, and α1D-AR, by molecular cloning and pharmacological analysis (Foglar et al. 1995; Guarino et al. 1996; Han et al. 1987; McGrath 1982). Since catecholamines stimulate vascular smooth muscle contraction by activating α1-ARs, α1-AR antagonists were originally introduced for the treatment of hypertension. Following the publication of clinical trials, such as Antihypertensive and Lipid-Lowering Treatment ⁎ Corresponding author. Department of Pharmacology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan. E-mail address:
[email protected] (A. Sanbe). 1 Authors contributed equally to this study. 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.02.020
to Prevent Heart Attack (ALLHAT) (2000) and V-HeFT (Cohn 1988), it was revealed that the α1-AR antagonists are less effective than other antihypertensive drugs such as thiazide-type diuretics and angiotensin-converting enzyme inhibitors, while blood pressure is significantly reduced from entry levels and remains lower (Krakoff 2001). Thus, α1AR antagonists are no longer considered as first-line drugs for this indication (Chobanian et al. 2003). In contrast to anti-hypertension, α1-AR antagonists are extensively used in the treatment of lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) (van Dijk et al. 2006). For the treatment of BPH, vasodilating action is regarded as a side effect of the α1-AR antagonists. Additionally, there was a tendency for stroke as well as angina, and a definite tendency for heart failure to occur more often in the doxazosin group than in the group taking chlorthalidone in the ALLHAT study, although the underlying mechanism explaining the poor outcome of the α1-AR antagonists has not been fully elucidated (Krakoff 2001). These results imply that the long-term blockade of the α1-AR may lead to a disturbance in the vascular system that may trigger adverse incidences in clinical cases. Thus, characterization of the long-term effect of α1-AR blockade on vasculatures is clinically important.
714
A. Sanbe et al. / Life Sciences 84 (2009) 713–718
Fig. 1. Body weight (A), systolic blood pressure (B) and heart rate (HR) (C) in conscious WT and α1-AR triple-KO mice. Blood pressure and heart rate in conscious condition were determined by the tail-cuff method as described in Materials and methods. Reduced blood pressure is seen in α1-AR triple-KO mice; n = 6 for WT and n = 6 for α1-AR triple-KO mice in each experiment. ⁎⁎⁎P b 0.001 vs. WT mice.
For the lack of α1-AR subtype-specific antagonists, α1-AR subtypedeficient mice generated by gene targeting represent a powerful tool for analysis of the functional roles of individual subtypes. Our previous studies have recognized the contributions of each α1-AR subtype to NA-induced vasoconstrictive responses and they have been characterized by others using mice with targeted disruption of each α1-AR subtype gene (Cavalli et al. 1997; Hosoda et al. 2005a, 2007; Rokosh and Simpson 2002; Sanbe et al. 2007; Tanoue et al. 2002). In addition, we recently demonstrated that all α1-AR subtype-deficient mice (α1AR triple-KO mice) show a lack of in vitro responsiveness of vasoconstriction induced by NA and loss of in vivo pressor response against phenylephrine injection (non-selective α1-AR agonist) (Sanbe et al. 2007). This α1-AR triple-KO mouse can be a useful model for long-term treatment with α1-AR blockers. In the present study, we have characterized the vascular responsiveness to two vasoconstrictors such as PGF2α and 5-HT in α1-AR triple-KO mice.
C57BL/6 mouse six times to maintain a C57BL/6 background. To generate all α1-AR subtype KO (α1-AR triple KO) mice, each α1-AR KO mouse with a C57BL/6 background was crossbred as described previously (Sanbe et al. 2007). α1-AR triple-KO mice could survive, develop normally, and grow for at least 1 year, and similar tissue weight was observed between α1-AR triple-KO and wild-type (WT) mice as described previously (Sanbe et al. 2007). All mice were housed in micro-isolator cages in a pathogen-free barrier facility and placed on a 12/12-hr light/dark cycle with ad libitum access to food and water. All data presented here were obtained from male mice aged 8– 10 weeks. All experiments were performed under the approved institutional guidelines for the Care and Use of Laboratory Animals of the National Research Institute for Child Health and Development.
Materials and methods Gene-targeted mice Each subtype of α1-AR KO mice, α1A-AR, α1B-AR, and α1D-AR KO mice was generated by gene targeting (Cavalli et al. 1997; Rokosh and Simpson 2002; Tanoue et al. 2002), and was backcrossed with a
Fig. 2. High potassium-induced vascular smooth muscle contraction in the aortic segments of WT and α1-AR triple-KO mice. The concentration of potassium was 80 mM (80 K) and 20 mM (20 K). (1) and (2) in 80 mM potassium stimulation correspond to the first and second contraction; n = 6 for WT and n = 6 for α1-AR triple-KO mice in each experiment. Submaximal contraction by potassium (20 K) was enhanced in α1-AR triple-KO mice. ⁎⁎P b 0.01 vs. WT mice.
Fig. 3. PGF2α- and 5-HT-induced vascular smooth muscle contraction in the aortic segments of WT and α1-AR triple-KO mice. (A) and (B) show the concentration–response curve of contraction stimulated by 10− 8 to 3 × 10− 5 M of PGF2α and 10− 9 to 10− 5 M of 5HT, respectively; n = 6 for both WT and α1-AR triple-KO mice in each experiment. Contractile response to PGF2α and 5-HT in α1-AR triple-KO mice was shifted leftward of that of WT mice. ⁎P b 0.05, ⁎⁎P b 0.01 vs. WT mice.
A. Sanbe et al. / Life Sciences 84 (2009) 713–718
715
Table 1 pD2 and maximum tension values for several vasoconstrictors in the aorta. α1-AR triple KO
WT
NA PGF2α 5-HT
n
pD2
Max (mg/mg wet weight)
n
pD2
Max (mg/mg wet weight)
6 5 5
8.04 ± 0.17 5.42 ± 0.16 6.59 ± 0.17
114.1 ± 22.3 120.3 ± 25.5 128.0 ± 26.0
5 5 5
n.d. 5.94 ± 0.04⁎ 7.19 ± 0.03⁎
− 1.9 ± 0.8⁎⁎ 142.0 ± 5.2 119.9 ± 5.8
Values are mean ± SEM. * pb 0.05, ** pb 0.01 vs. WT.
Mechanical responses The responses of the thoracic aorta to stimulation with the vasoconstrictors were elicited as described previously (Hosoda et al. 2005a; Sanbe et al. 2007; Tanoue et al. 2002). Briefly, after anesthesia, the thoracic aorta was isolated and excess fat and connective tissue were removed. Isolated artery was cut helically into a section 1520 mm in length and 1 mm in width. The intimal surface of the artery was gently rubbed with moistened filter paper to remove the endothelium, and the functional absence of the endothelium was confirmed by the lack of relaxant response to acetylcholine (10 μM). Aortic preparations were suspended in a 20-ml organ bath filled with normal Tyrode's solution (NaCl, 158.3 mM; KCl, 4.0 mM; CaCl2, 2.0 mM; MgCl2, 1.05 mM; NaH2PO4, 0.42 mM; NaHCO3, 10.0 mM; glucose, 5.6 mM) kept at 36.5 ± 0.5 °C and bubbled with a mixture of 95% O2 and 5% CO2. To prevent the oxidation of NA, L-ascorbic acid (10 μM) was added to the solution. The tension was monitored continuously and was recorded isometrically by a force displacement transducer. Experiments were performed in the presence of propranolol (1 μM) and yohimbine (0.3 μM) to block β1/β2-ARs and α2-ARs, respectively, and desipramine (0.3 μM) and deoxycorticosterone acetate (10 μM) to inhibit the neural and non-neural uptake of NA, respectively. The aortic segment was allowed to equilibrate for 90 min under a resting tension of 0.5 g and then contracted with KCl, NA, PGF2α, and 5-HT. This was repeated until two successive contractions of approximately equal size had been obtained. The concentration– response curves of PGF2α and 5-HT were obtained cumulatively. Measurement of blood pressure and heart rate The systolic blood pressure and heart rate (HR) were measured in conscious mice with a computerized tail-cuff system (BA-98A system; Softron Co., Tokyo, Japan) that determines systolic blood pressure using a photoelectric sensor as described previously (Sanbe et al. 2007). After measurement, the mice were anesthetized with sodium pentobarbital (40 mg/kg i.p.). After anesthesia, a PE-10 polyethylene catheter (Clay Adams, Parsippany, NJ) was inserted into the right carotid artery. The catheter was connected to a pressure transducer
Fig. 5. Gene expression and protein levels of the hormone receptors by real-time RT-PCR (A) and Western blot analysis (B and C) in aorta from WT and α1-AR triple KO mice; n=6 for both WT and α1-AR triple KO mice in each experiment. Increased expression (A) and protein levels (B and C) of 5-HT2A were observed in the thoracic aorta from α1-AR triple KO mice. FP, FP receptor; 5-HT2A, 5-HT2A receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
(SPR-671, Millar Instruments, Houston, TX), and the mean arterial blood pressure (MAP) was recorded on a PowerLab system (Bio Research Center, Nagoya, Japan). Measurement of HR was triggered by changes in MAP (AT-601G; Nihon Kohden Corp, Tokyo, Japan). Propranolol (3 mg/kg) was injected before the experiments to prevent the effects of β-AR signaling. To examine the pressor responses, 30 μl of PGF2α was administered through a catheter inserted into the right femoral or jugular veins as a bolus at intervals of 15–30 min after ensuring that the MAP and HR had returned to the basal levels. Fig. 4. In vivo pressor response to PGF2α in WT and α1-AR triple-KO mice. The changes in mean arterial pressure (MAP) from basal levels are shown as delta MAP (mm Hg). Drugs were intravenously injected through the jugular vein, and the mean arterial pressure (MAP) was monitored as described in the Materials and methods section. The figure shows the concentration–response curve of blood pressure stimulated by 10–30 μg/kg of PGF2α; n = 8 for both WT and α1-AR triple-KO mice in each experiment. ⁎P b 0.05 vs. WT mice.
Isolation of total RNA and reverse transcription-polymerase chain reaction Mice were anesthetized with ether and sacrificed by cervical dislocation, and total RNA was isolated from aorta using Isogen
716
A. Sanbe et al. / Life Sciences 84 (2009) 713–718
(Nippon Gene, Tokyo, Japan). First-strand cDNA was synthesized from 5 μg of DNase-digested total RNA by SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) in a 20-μl reaction volume. The primers for reverse transcription-polymerase chain reaction (RTPCR) were designed as follows: 5′-TTCTGCTCCGGACACAACCACTC-3′ upstream and 5′-AAGCTGTCCCCTCAAGTCATC-3′ downstream for the FP receptor, 5′-TTTCCTTGTCATGCCCGTGTC-3′ upstream and 5′AAGAGCACATCCAGGTAAATCCAG-3′ downstream for the 5-HT2A receptor. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were 5′-CCATCACCATCTTCCAGGAG-3′ upstream and 5′TCCAGCTCTGGGATGACCTT-3′ downstream as a standard. A quantitative real-time PCR assay was performed with the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq containing SYBR Green I. We used the GAPDH gene to normalize the cDNA in order to quantify gene expression levels among the samples. The primers were used as described above. The reactions were prepared in reaction volumes of 20 μl and cycle-sequencing reactions were performed for 1 cycle of 10 s at 95 °C and 40 amplification cycles of 5 s at 95 °C and 30 s at 60 °C. Western blot analysis Mice were anesthetized with ether and sacrificed by cervical dislocation, and total protein was isolated from aorta as described previously (Sanbe et al. 2003). Western blot analyses were performed with the use of anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon International, Temecula, CA), anti-5-HT2A receptor (ab16028; Abcam Inc., Cambridge, MA) and anti-FR receptor antibody (Cayman, Ann Arbor, MI). The band intensity in the immunoblot was semi-quantified using Image J1. Statistical analyses Data are expressed as mean ± standard error. Statistical analysis was performed using the unpaired Student's t-test or ANOVA followed by a post-hoc comparison with Fisher's PLSD using Statview version 5.0 software (Concepts Inc., Berkeley, CA). Differences between groups were considered statistically significant at the level of P b 0.05. Apparent pD2 value (negative logarithm of 50% effective concentration) and maximum constriction were calculated from concentration– response curves constructed from the experiments. Results Blood pressure in conscious mice The α1-AR triple-KO mice showed a reduced level of conscious blood pressure as compared with WT mice (Fig.1B). There was no difference in body weight or heart rate between the two groups (Fig. 1A and C). Vascular contractility in isolated thoracic aorta We examined the contractile function of the thoracic aorta in response to a high concentration of KCl (20 or 80 mM) (Fig. 2). The maximum developed tensions in response to depolarization stimulus with 80 mM KCl were 87.4 ± 13.4 mg/mg wet tissue (n = 6) (first contraction), 108.1 ± 16.7 mg/mg wet tissue (n = 6) (second contraction) in WT, and 86.5 ± 12.7 mg/mg wet tissue (n = 5) (first contraction), 105.5 ± 12.4 mg/mg wet tissue (n = 5) (second contraction) in α1-AR triple-KO mice (Fig. 2). There was no significant difference in the 80 mM KCl-induced vasoconstriction between WT and α1-AR triple-KO mice [P = 0.96 (first contraction) and 0.91 (second contraction) for WT vs. α1-AR triple-KO mice by the Student's t-test]. In contrast to the maximum stimulus, enhanced phasic constriction followed by a tonic constriction was observed by sub-maximal stimulus with 20 mM KCl in α1-AR triple-KO mice
compared with that in WT mice (P = 0.005 and P = 0.07 for WT vs. α1-AR triple-KO mice in phasic and tonic constrictions, respectively, by the Student's t-test) (Fig. 2). In addition, the contractile tension was markedly developed by the vasopressor agents PGF2α and 5-HT at lower concentrations, and the concentration–response curves were shifted toward the left in α1-AR triple-KO mice compared with WT mice by a factor of 3.3 and 4.0 for PGF2α and 5-HT, respectively (Fig. 3). The pD2 values and maximum tensions calculated by the concentration–response curves are summarized in Table 1. No NAinduced contraction was observed in α1-AR triple-KO mice, consistent with previous data (Sanbe et al. 2007). Although no significant difference was observed in either PGF2α- or 5-HT-induced maximum tensions between WT and α1-AR triple-KO mice (P = 0.45 and P = 0.78 for WT vs. α1-AR triple-KO mice in response to PGF2α and 5-HT, respectively, by the Student's t-test), pD2 values in α1-AR triple-KO mice were significantly lower than those in WT mice (Table 1). Blood pressure measurements in response to PGF2α We injected PGF2α intravenously into mice to examine the druginduced pressor responses. The in vivo pressor response induced by PGF2α at 30 and 100 μg/kg was enhanced in α1-AR triple-KO mice compared with that in WT mice (P b 0.001 in WT vs. α1-AR triple-KO mice by two-way ANOVA and P b 0.05 in WT vs. α1-AR triple-KO mice by Fisher's PLSD) (Fig. 4). No significant difference in the heart rate response was observed between WT and α1-AR triple-KO mice during the experiments (data not shown). We also tried to determine in vivo pressor response to 5-HT in the α1-AR triple-KO and WT mice in this study. Since 5-HT has multiple effects on blood pressure such as a depressor effect which is generated by the von Bezold–Jarisch reflex via the 5-HT3 receptor and a pressor effect which is mediated by contraction of arteries via 5-HT2A (Yamano et al. 1995), quantitative characterization of in vivo contractile response to 5-HT was difficult in the present study. Gene expression and protein levels of the receptors Since α1-AR triple-KO mice showed enhanced responsiveness to PGF2α and 5-HT, we analyzed the gene expression as well as protein levels of these receptors, since RT-PCR indicates that they may be involved in arterial drug responsiveness (Koshimizu et al. 2006; McKune and Watts 2001; Ponicke et al. 2000). The gene expression as well as the protein level of the PGF2α receptor, FP receptor, in α1-AR triple-KO mice was similar to that in WT mice (Fig. 5A–C). In contrast, the expression and protein levels of the 5-HT receptor, 5-HT2A, were 1.5-fold higher in thoracic aorta from α1-AR triple-KO mice (Fig. 5A–C). Discussion In the present study, we demonstrate that the aorta of α1-AR triple-KO mice is highly sensitive to contractile stimulus with high potassium, PGF2α and 5-HT, while the specific binding activity of the α1-AR agonist and the contractile response to NA are lost completely in the aorta of α1-AR triple-KO mice (Sanbe et al. 2007). These results suggest that loss of α1-ARs can lead to enhancement of vascular responsiveness to vasoconstrictors other than NA, and imply that α1ARs and the subsequent signaling could be involved in regulating the vascular responsiveness to other stimulation such as depolarization and 5-HT. In our previous studies, we analyzed the in vitro contractile response of the aorta as well as in vivo pressor response of MAP in the α1B-AR KO, α1D-AR KO, and α1BD-AR double KO mice (Hosoda et al. 2005a,b), and showed that the increase in sensitivity of responsiveness was barely seen in the aorta from the α1BD-AR double KO mice (Hosoda et al. 2005a,b), although blood pressure is known to be similar between α1-AR triple-KO and α1BD-AR double KO mice (Sanbe et al. 2007). Thus, it is unlikely that this enhanced sensitivity is
A. Sanbe et al. / Life Sciences 84 (2009) 713–718
associated with the level of systemic blood pressure. In addition, all α1-AR blockades may be required for enhanced vascular contractility. A similar phenomenon has been observed in our previous study (Hosoda et al. 2007). Single deletion of α1A-AR or α1B-AR subtype genes does not inhibit the neointimal formation after vascular injury in the femoral artery, while deletion of both the α1A-AR and α1B-AR subtype genes significantly inhibits it (Hosoda et al. 2007). These past and present observations suggest that each α1-AR can cooperate and compensate the receptor function when the other receptor is absent (Hosoda et al. 2007; Sanbe et al. 2007). The enhanced vascular sensitivity was also dissociated with increase in expression levels of agonist receptors; the expression and protein levels of the PGF2α receptor, FP receptor, in α1-AR tripleKO mice were similar to those in WT mice, whereas the expression and protein levels of 5-HT receptor, 5-HT2A, were increased in α1-AR triple-KO mice compared with those in WT mice. Similar findings have been observed in chronic reserpine-treated animals in which endogenous catecholamine is depleted (Fleming et al. 1973; Insel 1989; Taki et al. 2004). Treatment with reserpine causes hypersensitivity in the arterial contractile responses to 5-HT and KCl, resulting in the leftward shift of concentration–response curves (Fleming et al. 1973; Insel 1989; Taki et al. 2004). The enhanced vascular sensitivity in chronic reserpine-treated animals was also dissociated with increase in expression levels of agonist receptors (Fleming et al. 1973; Insel 1989; Taki et al. 2004). Since a phenotype, such as enhanced sensitivity of vascular contractile response to agonists, observed in a catecholamine-depleted animal is similar to that in an α1-AR-deficient animal (Fleming et al. 1973; Insel 1989; Taki et al. 2004), this may imply that the deficiency of α1-adrenergic signaling due to deficiency of α1-AR as well as catecholamine depletion results in hypersensitivity against other agonist stimuli. Since no consistent change in receptor expression was seen in α1-AR-deficient mice, the α1-AR signaling may regulate sensitivity of the other receptors and/or downstream signaling such as membrane depolarization, mobilization of calcium, and alterations in adenylate cyclase in addition to enhanced receptor expression, although the possible involvement of nonspecific supersensitivity cannot be ruled out (Fleming et al. 1973; Insel 1989; Taki et al. 2004). Publication of clinical trials, such as ALLHAT (2000) and V-HeFT (Cohn 1988), clarified that the α1-AR antagonists are less effective than other antihypertensive drugs (Krakoff 2001). There was a tendency for stroke as well as angina, and a definite tendency for heart failure to occur more often in the α1-AR antagonist group than in the other groups (Krakoff 2001), even though α1-AR antagonists improve a patient's metabolic profile by raising high-density lipoprotein levels, lowering triacylglycerol levels, and increasing sensitivity to insulin (Andersen et al. 1998; Hooper 2001). These antagonists also improve fibrinolysis, as evidenced by lower plasminogen activator inhibitor-1 (PAI-1) activity and higher tissue plasminogen activator (tPA) activity (Andersen et al. 1998). These previous studies imply that the mechanism explaining the poor outcome of the clinical study on the α1-AR antagonists is certainly unknown. Thus, our present results strongly suggest that the α1-ARs can regulate vascular responsiveness to the vasoconstrictors and blockade of the α1-ARs, and that subsequent signaling may enhance the vascular contractility in response to stimuli other than NA. This alteration may adversely affect the outcome of stroke and angina as well as heart failure. Further studies are needed to examine the longterm blockade of α1-AR signaling in the cardiovascular system to analyze the mechanism of adverse effect of α1-AR antagonists by long-term use. In conclusion, our results reveal enhancement of vascular contractility in response to vasoconstrictors such as high potassium, PGF2α and 5-HT in α1-AR triple-KO mice, which lacks the three α1-AR subtypes, α1A-AR, α1B-AR, and α1D-AR. These results suggest that the three α1-AR subtypes may play an important role in regulation of
717
other vasoconstricting signals by suppressing their receptor expressions. This phenomenon may be a reason for the poor outcome in clinical trials for cardiovascular disease using α1-AR antagonists. Acknowledgments This work was supported in part by research grants from the Scientific Fund of the Ministry of Education, Culture, Sports, Science and Technology of Japan, Ministry of Health, Labour and Welfare of Japan, the Japan Health Sciences Foundation, Novartis Foundation, Suzuken Memorial Foundation, and Japan Heart Foundation/Novartis Grant for Research Award on Molecular and Cellular Cardiology, Takeda Science Foundation, and Mochida Memorial Foundation for Medical and Pharmaceutical Research. References ALLHAT Collaborative Research Group. Major cardiovascular events in hypertensive patients randomized to doxazosin vs chlorthalidone: The antihypertensive and lipid-lowering treatment to prevent heart attack trial (ALLHAT). The Journal of the American Medical Association 283 (15), 1967–1975, 2000. Andersen P, Seljeflot I, Herzog A, Arnesen H, Hjermann I, Holme I. Effects of doxazosin and atenolol on atherothrombogenic risk profile in hypertensive middle-aged men. Journal of Cardiovascular Pharmacology 31 (5), 677–683, 1998. Burcelin R, Uldry M, Foretz M, Perrin C, Dacosta A, Nenniger-Tosato M, Seydoux J, Cotecchia S, Thorens B. Impaired glucose homeostasis in mice lacking the alpha1badrenergic receptor subtype. Journal of Biological Chemistry 279 (2), 1108–1115, 2004. Cavalli A, Lattion AL, Hummler E, Nenniger M, Pedrazzini T, Aubert JF, Michel MC, Yang M, Lembo G, Vecchione C, Mostardini M, Schmidt A, Beermann F, Cotecchia S. Decreased blood pressure response in mice deficient of the alpha1b-adrenergic receptor. Proceedings of the National Academy of Sciences 94 (21), 11589–11594, 1997. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo Jr JL, Jones DW, Materson BJ, Oparil S, Wright Jr JT, Roccella EJ. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: The report of the Joint National Committee 7 Report. The Journal of the American Medical Association 289 (19), 2560–2572, 2003. Cohn JN. Effect of vasodilator therapy on mortality in chronic congestive heart failure. European Heart Journal 9 Suppl A, 171–173, 1988. Fleming WW, McPhillips JJ, Westfall DP. Postjunctional supersensitivity and subsensitivity of excitable tissues to drugs. Ergebnisse der Physiologie, Biologischen Chemie und Experimentellen Pharmakologie 68, 55–119, 1973. Foglar R, Shibata K, Horie K, Hirasawa A, Tsujimoto G. Use of recombinant alpha 1adrenoceptors to characterize subtype selectivity of drugs for the treatment of prostatic hypertrophy. European Journal of Pharmacology 288 (2), 201–207, 1995. Guarino RD, Perez DM, Piascik MT. Recent advances in the molecular pharmacology of the alpha 1-adrenergic receptors. Cellular Signalling 8 (5), 323–333, 1996. Han C, Abel PW, Minneman KP. Alpha 1-adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle. Nature 329 (6137), 333–335, 1987. Hooper PL. Reduced heat shock proteins: A mechanism to explain higher cardiovascular events associated with doxazosin. Journal of Human Hypertension 15 (4), 285, 2001. Hosoda C, Koshimizu TA, Tanoue A, Nasa Y, Oikawa R, Tomabechi T, Fukuda S, Shinoura H, Oshikawa S, Takeo S, Kitamura T, Cotecchia S, Tsujimoto Ga. Two alpha1adrenergic receptor subtypes regulating the vasopressor response have differential roles in blood pressure regulation. Molecular Pharmacology 67 (3), 912–922, 2005a. Hosoda C, Tanoue A, Shibano M, Tanaka Y, Hiroyama M, Koshimizu TA, Cotecchia S, Kitamura T, Tsujimoto G, Koike Kb. Correlation between vasoconstrictor roles and mRNA expression of alpha1-adrenoceptor subtypes in blood vessels of genetically engineered mice. British Journal of Pharmacology 146 (3), 456–466, 2005b. Hosoda C, Hiroyama M, Sanbe A, Birumachi J, Kitamura T, Cotecchia S, Simpson PC, Tsujimoto G, Tanoue A. Blockade of both alpha1A- and alpha1B-adrenergic receptor subtype signaling is required to inhibit neointimal formation in the mouse femoral artery. The American Journal of Physiology. Heart and Circulatory Physiology 293 (1), H514–H519, 2007. Insel PA. Structure and function of alpha-adrenergic receptors. The American Journal of Medicine 87 (2A), 12S–18S, 1989. Koshimizu TA, Kretschmannova K, He ML, Ueno S, Tanoue A, Yanagihara N, Stojilkovic SS, Tsujimoto G. Carboxyl-terminal splicing enhances physical interactions between the cytoplasmic tails of purinergic P2X receptors. Molecular Pharmacology 69 (5), 1588–1598, 2006. Krakoff LR. Comments on ALLHAT and doxazosin. Current Controlled Trials in Cardiovascular Medicine 2 (6), 254–256, 2001. McGrath JC. Evidence for more than one type of post-junctional alpha-adrenoceptor. Biochemical Pharmacology 31 (4), 467–484, 1982. McKune CM, Watts SW. Characterization of the serotonin receptor mediating contraction in the mouse thoracic aorta and signal pathway coupling. The Journal of Pharmacology and Experimental Therapeutics 297 (1), 88–95, 2001.
718
A. Sanbe et al. / Life Sciences 84 (2009) 713–718
O'Connell TD, Ishizaka S, Nakamura A, Swigart PM, Rodrigo MC, Simpson GL, Cotecchia S, Rokosh DG, Grossman W, Foster E, Simpson PC. The alpha(1A/C)- and alpha(1B)adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse. The Journal of Clinical Investigation 111 (11), 1783–1791, 2003. Ponicke K, Giessler C, Grapow M, Heinroth-Hoffmann I, Becker K, Osten B, Brodde OE. FP-receptor mediated trophic effects of prostanoids in rat ventricular cardiomyocytes. British Journal of Pharmacology 129 (8), 1723–1731, 2000. Rokosh DG, Simpson PC. Knockout of the alpha 1A/C-adrenergic receptor subtype: The alpha 1A/C is expressed in resistance arteries and is required to maintain arterial blood pressure. The Proceedings of the National Academy of Sciences 99 (14), 9474–9479, 2002. Sanbe A, Gulick J, Hanks MC, Liang Q, Osinska H, Robbins J. Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter. Circulation Research 92 (6), 609–616, 2003. Sanbe A, Tanaka Y, Fujiwara Y, Tsumura H, Yamauchi J, Cotecchia S, Koike K, Tsujimoto G, Tanoue A. Alpha1-adrenoceptors are required for normal male sexual function. British Journal of Pharmacology 152 (3), 332–340, 2007.
Taki N, Tanaka T, Zhang L, Suzuki F, Israilova M, Taniguchi T, Hiraizumi-Hiraoka Y, Shinozuka K, Kunitomo M, Muramatsu I. Alpha-1D adrenoceptors are involved in reserpine-induced supersensitivity of rat tail artery. British Journal of Pharmacology 142 (4), 647–656, 2004. Tanoue A, Nasa Y, Koshimizu T, Shinoura H, Oshikawa S, Kawai T, Sunada S, Takeo S, Tsujimoto G. The alpha(1D)-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction. The Journal of Clinical Investigation 109 (6), 765–775, 2002. van Dijk MM, de la Rosette JJ, Michel MC. Effects of alpha(1)-adrenoceptor antagonists on male sexual function. Drugs 66 (3), 287–301, 2006. Yamano M, Ito H, Kamato T, Miyata K. Species difference in the 5-hydroxytryptamine3 receptor associated with the von Bezold–Jarisch reflex. Archives Internationales de Pharmacodynamie et de Thérapie 330 (2), 177–189, 1995.
